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361 FRA1 as the regulator of psoriasis-associated hyperproliferation and EMT transition of keratinocytes
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In vitro models of infant skin to study molecular mechanisms involved in pediatric atopic dermatitis S Bredif, G Belleme`re, F Menu and C Baudouin Laboratoires Expanscience, EPERNON, France AD pathogenesis is complex and involves multiple parameters. It is widely accepted that a Th2 dysbalance is a predominant part of AD pathogenesis. In order to study the molecular mechanisms involved in the development of AD in infants, we developed two complementary models of infant epidermis, one mimicking the Th2 induction phase and one reproducing the specific Th2-inflammatory state. RHE from a 6 months old donor have been stimulated by poly(I:C)+IL1-alpha (model of Th2 induction) or a mix of Th2 cytokines (IL4+IL13+IL22+TNF-alpha, model of Th2 inflammatory environment). Poly(I:C)+IL1 induced an overexpression of IL18, a cytokine involved in Th2 predominant inflammation, as well as the chemokines CCL3, CCL5 and CCL7 known to play an important role in the recruitment of inflammatory cells in AD, particularly Th2 cells, showing that this model actually mimicked early step of Th2 inflammation induction. Poly(I:C)+IL1 also induced an overexpression of KLK5, which activity is increased in AD and may contribute to exacerbation of barrier defect and itch. Th2 cytokines mix induced a strong decrease of keratinocyte differentiation and barrier markers and increased the expression of pro-inflammatory cytokines. These results show that this model was able to mimick the Th2 inflammation and barrier alteration representing main features of AD. Moreover, the Th2 cytokines repressed stem cells markers expression in this model. To our knowledge, this is the first observation of an impact of AD Th2-linked pathogenesis on stem cells pool. We used these models to evaluate a lipidreplenishing balm specifically formulated for baby and child atopic skin. In both model, the balm was able to counteract the effects of the stress and thus could efficiently modulate the impact of Th2 inflammation in AD. These two infant skin models of AD reproduce the induction and the maintenance of Th2-inflammation, and mimick the main features observed in AD. A possible impact of Th2-inflammatory environment on infant stem cells pool, which is particularly vulnerable to external aggression, was also observed.
Regulatory T cells in atopic dermatitis: Immune-suppressive or pro-inflammatory? V Moosbrugger-Martinz1, R Gruber2, K Ladsta¨tter1, M Bellutti1, M Schmuth1 and S Dubrac1 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 2 Department of Human Genetic, Medical University of Innsbruck, Innsbruck, Austria Although the important role of abnormal immune reactivity in atopic dermatitis (AD) is widely accepted, the complex network of the AD immune system remains still unclear, especially when it comes down to the role of regulatory T cells (Tregs). The heterogeneity of the AD population is certainly responsible for the absence of a clear view on the implication of Tregs in AD, despite several attempts by other research groups. We here describe the phenotype of blood Tregs in an AD population stratified by filaggrin loss-of function mutations and EASI score. Preliminary results show consistent increased in percentages of Tregs in the blood of AD patients associated with increased ICOS expression, regardless of EASI score. Within the Treg population, proportions of effector CCR4+CD45RAlow Tregs are enhanced, in contrast to proportions of naı¨ve and recently thymus emigrated Tregs. The Treg population exhibits a large plasticity and Tregs can turn into Th2, Th1 or Th17 like cells. We here show that proportions of Th2 Tregs are increased whereas proportions of Th1 Tregs are decreased in patients with AD, regardless of EASI score. Both Tregs and CD4+ non Treg lymphocytes isolated from AD patients produce more IL-10. IL-10 can suppress Th1 and Th2 immune responses but also promote Th2 immune response and mast cell activation. Thus, it remains to identify which of this role IL-10 plays in AD to be able to delineate the exact role of Tregs in the disease pathogenesis or maintenance.
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Thymic stromal lymphopoietin is a trigger for CD4+ T cell migration in atopic dermatitis L Wallmeyer1, K Dietert1, M Sochorova1, A Gruber1, K Vavrova2 and S Hedtrich1 1 Freie Universita¨t Berlin, Berlin, Germany and 2 Charles University Prague, Prague, Czech Republic Loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing factor for atopic dermatitis (AD), but also immunologic mechanisms are involved in its pathophysiology. The release of cytokines and cellular crosstalk between keratinocytes and lymphocytes are important promoters for the pathogenesis of AD. Moreover, thymic stromal lymphopoietin (TSLP) is highly expressed by keratinocytes in lesional skin of AD patients and stimulates the differentiation of naı¨ve CD4+ T cells into Th2 cells via activation of dendritic cells, which contribute to the induction of inflammatory responses. Aiming for a more detailed understanding of the interplay between CD4+ T cells and filaggrin-deficient skin, a FLG knock down skin model was used which is characterized by increased TSLP levels. Naı¨ve CD4+ T cells were isolated from human peripheral blood mononuclear cells by negative selection, activated with anti-CD3/CD28-beads and thereafter, added to the skin models. Interestingly, migration of CD4+ T cells into the dermal equivalent of the skin models was exclusively observed in filaggrin-deficient skin but not in normal skin models. Notably, T cells preincubated with a selective TSLP receptor antibody were not able to migrate. In contrast, direct application of recombinant human TSLP on dermis equivalents resulted in successful T cell migration, indicating that TSLP plays a potential role for the migration of T cells into dermal tissue. As there are no dendritic cells implemented in the skin model used in this study, our data suggest a direct link between TSLP and stimulation of T cells without involvement of dendritic cells.
B-cells transcriptome analysis in pemphigus patients after anti-CD20 treatment F Caillot1, C Derambure2, N Berkani1, G Riou2, M Maho Vaillant1, S Calbo2, P Joly1 and P Musette1 1 Department of Dermatology, Inserm Unit 905, Charles Nicolle University Hospital, Rouen, France and 2 Inserm Unit 905, Normandy University, Rouen, France Pemphigus is a B-cell-mediated autoimmune disease affecting skin and mucous membranes. Pathogenic autoantibodies are directed against desmogleins, which are keratinocyte adhesion proteins. In a previous study, we have shown that B cell-depletion by an anti-CD20 antibody (Mabthera) is able to induce a long-term complete remission in 59% of severe pemphigus patients after a 79-months median follow-up time. We aimed to understand the immunological mechanisms that mediate this long-lasting remission. We used fluorescence-assisted cell sorting to isolate B cells. We compared gene expression profile of B-cells from 3 patients who achieved complete remission off therapy and 3 who had incomplete remission or relapse, 79 months after initial Mabthera treatment and after restoration of B cells repertoire. We compared genome-wide expression profile by 4 44K whole human genome microarray, Agilent. GeneSpring GX software was used to determine mRNAs differentially regulated between complete and incomplete remission patients by t-test. To validate gene expression differences, some genes were assessed by qRT-PCR (QuantiGeneÒ Plex Assay technology) for a second set of patients (18 complete remissions and 7 incomplete remissions). Five of the 9 mRNA tested were confirmed differently expressed: KCa3.1 potassium channel and three collagens 4A3, 4A4 and 18A1 were down-regulated in B-cells of patients who had complete remission; conversely, beta1,6 N-acetyl glucosaminyl transferase V was over-expressed in the same group of patients. Furthermore, expression level of these RNAs in incomplete remission patients was similar to that measured in newly diagnosed untreated pemphigus patients (n¼12). Highlighted genes may play a role in the pathogenesis of pemphigus, through the B cell activation state.
S222 Journal of Investigative Dermatology (2016), Volume 136
FRA1 as the regulator of psoriasis-associated hyperproliferation and EMT transition of keratinocytes A Zolotarenko, A Prelovskaya, EV Chekalin, ES Piruzian and S Bruskin Laboratory of functional genomics, Vavilov Institute of General Genetics RAS, Moscow, Russian Federation FRA1, a transcription factor from AP-1 superfamily, is an important regulator of cell fate. The expression patterns of FRA1 in skin vary depending on the epidermal layer and the differentiation state of keratinocytes, and our previous research had shown FRA1 to be overexpressed in all epidermal layers of lesional skin of patients with psoriasis. The hallmarks of the disease are altered immune profiles of skin, keratinocyte hyperproliferation and abnormal differentiation, as well as extensive remodeling of extracellular matrix and keratinocyte’s acquisition of mesenchymal transition phenotype. Keratinocytes seem to be important mediators of the disease, not only responsible for visible skin manifestations and structural changes, but also capable for production of vast majority of psoriasis-associated cytokines, chemokines and antimicrobial molecules. In order to evaluate the role of FRA1 regulation in psoriasis-associated phenotype of keratinocytes we have created HaCaT keratinocytes with inducible FRA1 overexpression. qPCR analysis of FRA1-overexpressing cells has shown increased expression of proteases MMP1, MMP2 and MMP12 (ECM remodeling), cytokines TNFa and IL-8 (inflammation), SLUG, FN1, SERPINE1 (mesenchymal markers), CK16 (proliferation) and decreased expression of CK10 (differentiation). The influence of FRA1 overexpression on cell migration and wound healing was evaluated by scratch assay that has shown enhanced capability of FRA1-overexpressing cells to migrate toward the scratch. Our results support the hypothesis that FRA1 is an important player in the developing of psoriasisassociated skin manifestations, contributing to the elevated cytokine production by keratinocytes. Besides, FRA1-associated MMP-mediated ECM remodeling itself could promote the acquisition of mesenchymal phenotype by keratinocytes leading to the shift of CK expression, activation of proliferation and migration of the cells in the course of the disease.
Interest of 18-b glycyrrhetinic acid and Ginkgo biloba extract to complement acne therapy B Teme1, N Ardiet1, B Cadars2, S Trompezinski1, S Weber1, S Callejon1, M Chavagnac-Bonneville2 and E Jourdan2 1 NAOS Les Laboratoires, Aix-en-Provence, France and 2 Direction scientifique Bioderma (NAOS), Lyon, France Acne is a chronic inflammatory dermatosis in which Th17 was recently discovered to be activated. The treatment prescribed may generate adverse effects such as discomfort and skin dehydration. The purpose of this study was to assess the interest of active compounds to complement therapeutic treatment of acne in inhibiting IL-17-induced inflammation and soothing the adverse effects induced. An in vitro test was performed to assess the effect of active compounds on IL-17-induced IL-8 synthesis. After a 24-hour incubation, immortalized human keratinocytes were treated for 1 hour with various concentrations of 18-b glycyrrhetinic acid (18b-GA) and Ginkgo biloba extract (GBE), then with TNF-a and IL-17 (n¼3). After a 24-hour incubation, IL-8 was quantified in supernatants by ELISA and titration was normalized to cell viability. An occlusive patch test was conducted to assess the ability of 18b-GA to prevent sodium lauryl sulphate (SLS)-induced redness. Epicutaneous patches with vehicle only, 1% SLS, or 1% SLS plus various concentrations of 18b-GA were applied for 18 hours on the volar forearm of 20 healthy volunteers. Cutaneous color (a* parameter) was measured at t0 and t24hrs using SpectrocolorimeterÒ CM700-d (MINOLTA). An increase in a* parameter means skin redness appeared. 18b-GA had no effect on cytokine-induced inflammation but GBE induced a significant dose-dependent decrease up to 97% of IL-8 synthesis. 18b-GA significantly decreased a* parameter up to 55%, which indicates it decreased SLS-induced redness. GBE blocked inflammatory cascade induced by IL-17 in keratinocytes. We showed 18b-GA’s ability to limit the appearance of erythema induced by the irritant SLS under occlusion. There is an interest in including these active compounds in an adjunctive cosmetic care in the therapeutic treatment of acne to improve treatment efficacy thanks to its anti-inflammatory action and its soothing properties to enhance patients’ compliance.
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In vitro models of infant skin to study molecular mechanisms involved in pediatric atopic dermatitis S Bredif, G Belleme`re, F Menu and C Baudouin Laboratoires Expanscience, EPERNON, France AD pathogenesis is complex and involves multiple parameters. It is widely accepted that a Th2 dysbalance is a predominant part of AD pathogenesis. In order to study the molecular mechanisms involved in the development of AD in infants, we developed two complementary models of infant epidermis, one mimicking the Th2 induction phase and one reproducing the specific Th2-inflammatory state. RHE from a 6 months old donor have been stimulated by poly(I:C)+IL1-alpha (model of Th2 induction) or a mix of Th2 cytokines (IL4+IL13+IL22+TNF-alpha, model of Th2 inflammatory environment). Poly(I:C)+IL1 induced an overexpression of IL18, a cytokine involved in Th2 predominant inflammation, as well as the chemokines CCL3, CCL5 and CCL7 known to play an important role in the recruitment of inflammatory cells in AD, particularly Th2 cells, showing that this model actually mimicked early step of Th2 inflammation induction. Poly(I:C)+IL1 also induced an overexpression of KLK5, which activity is increased in AD and may contribute to exacerbation of barrier defect and itch. Th2 cytokines mix induced a strong decrease of keratinocyte differentiation and barrier markers and increased the expression of pro-inflammatory cytokines. These results show that this model was able to mimick the Th2 inflammation and barrier alteration representing main features of AD. Moreover, the Th2 cytokines repressed stem cells markers expression in this model. To our knowledge, this is the first observation of an impact of AD Th2-linked pathogenesis on stem cells pool. We used these models to evaluate a lipidreplenishing balm specifically formulated for baby and child atopic skin. In both model, the balm was able to counteract the effects of the stress and thus could efficiently modulate the impact of Th2 inflammation in AD. These two infant skin models of AD reproduce the induction and the maintenance of Th2-inflammation, and mimick the main features observed in AD. A possible impact of Th2-inflammatory environment on infant stem cells pool, which is particularly vulnerable to external aggression, was also observed.
Regulatory T cells in atopic dermatitis: Immune-suppressive or pro-inflammatory? V Moosbrugger-Martinz1, R Gruber2, K Ladsta¨tter1, M Bellutti1, M Schmuth1 and S Dubrac1 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 2 Department of Human Genetic, Medical University of Innsbruck, Innsbruck, Austria Although the important role of abnormal immune reactivity in atopic dermatitis (AD) is widely accepted, the complex network of the AD immune system remains still unclear, especially when it comes down to the role of regulatory T cells (Tregs). The heterogeneity of the AD population is certainly responsible for the absence of a clear view on the implication of Tregs in AD, despite several attempts by other research groups. We here describe the phenotype of blood Tregs in an AD population stratified by filaggrin loss-of function mutations and EASI score. Preliminary results show consistent increased in percentages of Tregs in the blood of AD patients associated with increased ICOS expression, regardless of EASI score. Within the Treg population, proportions of effector CCR4+CD45RAlow Tregs are enhanced, in contrast to proportions of naı¨ve and recently thymus emigrated Tregs. The Treg population exhibits a large plasticity and Tregs can turn into Th2, Th1 or Th17 like cells. We here show that proportions of Th2 Tregs are increased whereas proportions of Th1 Tregs are decreased in patients with AD, regardless of EASI score. Both Tregs and CD4+ non Treg lymphocytes isolated from AD patients produce more IL-10. IL-10 can suppress Th1 and Th2 immune responses but also promote Th2 immune response and mast cell activation. Thus, it remains to identify which of this role IL-10 plays in AD to be able to delineate the exact role of Tregs in the disease pathogenesis or maintenance.
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Thymic stromal lymphopoietin is a trigger for CD4+ T cell migration in atopic dermatitis L Wallmeyer1, K Dietert1, M Sochorova1, A Gruber1, K Vavrova2 and S Hedtrich1 1 Freie Universita¨t Berlin, Berlin, Germany and 2 Charles University Prague, Prague, Czech Republic Loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing factor for atopic dermatitis (AD), but also immunologic mechanisms are involved in its pathophysiology. The release of cytokines and cellular crosstalk between keratinocytes and lymphocytes are important promoters for the pathogenesis of AD. Moreover, thymic stromal lymphopoietin (TSLP) is highly expressed by keratinocytes in lesional skin of AD patients and stimulates the differentiation of naı¨ve CD4+ T cells into Th2 cells via activation of dendritic cells, which contribute to the induction of inflammatory responses. Aiming for a more detailed understanding of the interplay between CD4+ T cells and filaggrin-deficient skin, a FLG knock down skin model was used which is characterized by increased TSLP levels. Naı¨ve CD4+ T cells were isolated from human peripheral blood mononuclear cells by negative selection, activated with anti-CD3/CD28-beads and thereafter, added to the skin models. Interestingly, migration of CD4+ T cells into the dermal equivalent of the skin models was exclusively observed in filaggrin-deficient skin but not in normal skin models. Notably, T cells preincubated with a selective TSLP receptor antibody were not able to migrate. In contrast, direct application of recombinant human TSLP on dermis equivalents resulted in successful T cell migration, indicating that TSLP plays a potential role for the migration of T cells into dermal tissue. As there are no dendritic cells implemented in the skin model used in this study, our data suggest a direct link between TSLP and stimulation of T cells without involvement of dendritic cells.
B-cells transcriptome analysis in pemphigus patients after anti-CD20 treatment F Caillot1, C Derambure2, N Berkani1, G Riou2, M Maho Vaillant1, S Calbo2, P Joly1 and P Musette1 1 Department of Dermatology, Inserm Unit 905, Charles Nicolle University Hospital, Rouen, France and 2 Inserm Unit 905, Normandy University, Rouen, France Pemphigus is a B-cell-mediated autoimmune disease affecting skin and mucous membranes. Pathogenic autoantibodies are directed against desmogleins, which are keratinocyte adhesion proteins. In a previous study, we have shown that B cell-depletion by an anti-CD20 antibody (Mabthera) is able to induce a long-term complete remission in 59% of severe pemphigus patients after a 79-months median follow-up time. We aimed to understand the immunological mechanisms that mediate this long-lasting remission. We used fluorescence-assisted cell sorting to isolate B cells. We compared gene expression profile of B-cells from 3 patients who achieved complete remission off therapy and 3 who had incomplete remission or relapse, 79 months after initial Mabthera treatment and after restoration of B cells repertoire. We compared genome-wide expression profile by 4 44K whole human genome microarray, Agilent. GeneSpring GX software was used to determine mRNAs differentially regulated between complete and incomplete remission patients by t-test. To validate gene expression differences, some genes were assessed by qRT-PCR (QuantiGeneÒ Plex Assay technology) for a second set of patients (18 complete remissions and 7 incomplete remissions). Five of the 9 mRNA tested were confirmed differently expressed: KCa3.1 potassium channel and three collagens 4A3, 4A4 and 18A1 were down-regulated in B-cells of patients who had complete remission; conversely, beta1,6 N-acetyl glucosaminyl transferase V was over-expressed in the same group of patients. Furthermore, expression level of these RNAs in incomplete remission patients was similar to that measured in newly diagnosed untreated pemphigus patients (n¼12). Highlighted genes may play a role in the pathogenesis of pemphigus, through the B cell activation state.
S222 Journal of Investigative Dermatology (2016), Volume 136
FRA1 as the regulator of psoriasis-associated hyperproliferation and EMT transition of keratinocytes A Zolotarenko, A Prelovskaya, EV Chekalin, ES Piruzian and S Bruskin Laboratory of functional genomics, Vavilov Institute of General Genetics RAS, Moscow, Russian Federation FRA1, a transcription factor from AP-1 superfamily, is an important regulator of cell fate. The expression patterns of FRA1 in skin vary depending on the epidermal layer and the differentiation state of keratinocytes, and our previous research had shown FRA1 to be overexpressed in all epidermal layers of lesional skin of patients with psoriasis. The hallmarks of the disease are altered immune profiles of skin, keratinocyte hyperproliferation and abnormal differentiation, as well as extensive remodeling of extracellular matrix and keratinocyte’s acquisition of mesenchymal transition phenotype. Keratinocytes seem to be important mediators of the disease, not only responsible for visible skin manifestations and structural changes, but also capable for production of vast majority of psoriasis-associated cytokines, chemokines and antimicrobial molecules. In order to evaluate the role of FRA1 regulation in psoriasis-associated phenotype of keratinocytes we have created HaCaT keratinocytes with inducible FRA1 overexpression. qPCR analysis of FRA1-overexpressing cells has shown increased expression of proteases MMP1, MMP2 and MMP12 (ECM remodeling), cytokines TNFa and IL-8 (inflammation), SLUG, FN1, SERPINE1 (mesenchymal markers), CK16 (proliferation) and decreased expression of CK10 (differentiation). The influence of FRA1 overexpression on cell migration and wound healing was evaluated by scratch assay that has shown enhanced capability of FRA1-overexpressing cells to migrate toward the scratch. Our results support the hypothesis that FRA1 is an important player in the developing of psoriasisassociated skin manifestations, contributing to the elevated cytokine production by keratinocytes. Besides, FRA1-associated MMP-mediated ECM remodeling itself could promote the acquisition of mesenchymal phenotype by keratinocytes leading to the shift of CK expression, activation of proliferation and migration of the cells in the course of the disease.
Interest of 18-b glycyrrhetinic acid and Ginkgo biloba extract to complement acne therapy B Teme1, N Ardiet1, B Cadars2, S Trompezinski1, S Weber1, S Callejon1, M Chavagnac-Bonneville2 and E Jourdan2 1 NAOS Les Laboratoires, Aix-en-Provence, France and 2 Direction scientifique Bioderma (NAOS), Lyon, France Acne is a chronic inflammatory dermatosis in which Th17 was recently discovered to be activated. The treatment prescribed may generate adverse effects such as discomfort and skin dehydration. The purpose of this study was to assess the interest of active compounds to complement therapeutic treatment of acne in inhibiting IL-17-induced inflammation and soothing the adverse effects induced. An in vitro test was performed to assess the effect of active compounds on IL-17-induced IL-8 synthesis. After a 24-hour incubation, immortalized human keratinocytes were treated for 1 hour with various concentrations of 18-b glycyrrhetinic acid (18b-GA) and Ginkgo biloba extract (GBE), then with TNF-a and IL-17 (n¼3). After a 24-hour incubation, IL-8 was quantified in supernatants by ELISA and titration was normalized to cell viability. An occlusive patch test was conducted to assess the ability of 18b-GA to prevent sodium lauryl sulphate (SLS)-induced redness. Epicutaneous patches with vehicle only, 1% SLS, or 1% SLS plus various concentrations of 18b-GA were applied for 18 hours on the volar forearm of 20 healthy volunteers. Cutaneous color (a* parameter) was measured at t0 and t24hrs using SpectrocolorimeterÒ CM700-d (MINOLTA). An increase in a* parameter means skin redness appeared. 18b-GA had no effect on cytokine-induced inflammation but GBE induced a significant dose-dependent decrease up to 97% of IL-8 synthesis. 18b-GA significantly decreased a* parameter up to 55%, which indicates it decreased SLS-induced redness. GBE blocked inflammatory cascade induced by IL-17 in keratinocytes. We showed 18b-GA’s ability to limit the appearance of erythema induced by the irritant SLS under occlusion. There is an interest in including these active compounds in an adjunctive cosmetic care in the therapeutic treatment of acne to improve treatment efficacy thanks to its anti-inflammatory action and its soothing properties to enhance patients’ compliance.